There are currently many clinical trials for stem cell therapy underway worldwide that employ embryonic and adult stem cells. When using adult stem cells, the cells must first be isolated from the complex tissue of origin. The type of tissue from which adult stem cells can be isolated includes liver, heart, lung, adipose tissue, kidney, pancreas, bone marrow, and blood. Individual adult stem cells are isolated from complex tissue using fluorescence-activated cell sorting (FACS). FACS sorts cells, one cell at a time, based on the patterns of surface protein expression of each cell by incubating the cells with specific monoclonal antibodies that are conjugated to fluorescent molecules called fluorochromes. Theoretically, better results in clinical trials can be obtained by selection and administration of cells with certain surface protein expression patterns. The two main limitations of this selection method come from the fact that surface protein expression does not completely correlate with cell identity or function and that most groups of cells are heterogeneous in nature.
Gene expression profiling is a useful tool to define the identity and function of a group of cells, and can often provide more information than surface protein expression patterns alone. Selection of cells' for therapeutic use based on gene expression profiles offers significant advantages because this approach can define cell function more precisely. Methods of gene expression analysis include microarray, serial analysis of gene expression (SAGE), and polymerase chain reaction (PCR). More specifically, quantitative real time reverse transcriptase PCR (RT-PCR or qRT-PCR) is used to quantify the expression of a given gene or group of genes, which allows more precise comparison across samples. These methods are useful for defining the gene expression patterns of large populations of cells. In quantitative reverse transcriptase PCR, a messenger RNA strand is first reverse transcribed into cDNA, and then the resulting cDNA is amplified using real time/quantitative PCR (qPCR). This gives a quantitative assessment of the amount of RNA molecules for a specific gene within a cell or tissue as a measure of gene expression. Quantitative real time RT-PCR uses fluorescent probes that bind to DNA and allow for the visualization of the cDNA molecule as it is amplified during the PCR cycles. The point at which the probe becomes visible is precisely measured, which allows calculation of the amount of RNA from the gene of interest. The fluorescent signal produced from expression of each gene of interest is normalized against the signal from a reference gene measured in the same sample to account for possible variation in the amount and quality of RNA between different samples. Additionally, when many cells are analyzed as a single sample, the variances in gene expression among the cells cannot be analyzed because mRNA from all cells is pooled together. This population-averaging approach limits the ability of traditional analytic methods to define the gene expression patterns of a heterogeneous group of cells, such as stem cells.